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Han, Cogent Biology (2017), 3: 1336886
https://doi.org/10.1080/23312025.2017.1336886
IMMUNOLOGY | SHORT COMMUNICATION
In vitro biological activities of Douglas fir essential
oil in a human skin disease model
Xuesheng Han1*
Received: 21 March 2017
Accepted: 26 May 2017
Published: 05 July 2017
*Corresponding author: Xuesheng Han,
dōTERRA International, LLC, 389 S 1300
W, Pleasant Grove, UT 84062, USA
E-mails: [email protected], lawry.han@
gmail.com
Reviewing editor:
Maté Biro, University of New South
Wales, Australia
Additional information is available at
the end of the article
Abstract: Although essential oils from Douglas fir are popular topical skincare
products, research regarding their biological effects on human skin cells is scarce.
Here, we studied the biological activity of a commercially available Douglas fir
(Pseudotsuga menziesii) essential oil (DEO) in a human dermal fibroblast model
of chronic inflammation and fibrosis induced by stimulation with cytokines.
Chemical analysis of DEO indicated that its major chemical components (i.e.
>5%) were beta-pinene (23%), sabinene (17%), terpinolene (14%), delta-3-carene
(11%), and alpha-pinene (9%). We analyzed the effect of DEO on the levels of
17 important protein biomarkers associated with inflammation, immune system
modulation, and tissue remodeling. DEO exhibited significant anti-proliferative
activity in human fibroblasts. DEO also significantly inhibited the production
of vascular cell adhesion molecule 1, collagen III, and plasminogen activator
inhibitor 1. We also observed that DEO robustly modulated global gene expression levels in diverse ways. In particular, DEO affected the expression of genes
involved in immune modulation and cancer signaling. This study provides the first
evidence of biological activity of DEO in human dermal fibroblasts. Our results
suggest that DEO may modulate immune responses and tumor signaling processes. Further research about the biological and pharmacological mechanisms
of DEO action is recommended.
ABOUT THE AUTHORS
PUBLIC INTEREST STATEMENT
Han's group is specifically interested in the
efficacy and safety of essential oils and their
active components. Our studies of essential
oils in both in vitro and clinical settings utilize a
variety of experimental approaches, including
analytical, biological, biochemical, and
biomedical methodologies. The research work
discussed in this paper represents one part of a
large research project, which was designed to
extensively examine the impact of essential oils
on human cells. This study, along with others,
will further the understanding of the health
benefits of essential oils for a wide research
audience. Besides essential oils, we are also
interested in studying the health benefits of
herbal supplements and skin care products.
Han holds a PhD in Biological Sciences and is
an elected Fellow of the American College of
Nutrition.
Essential oils are popular worldwide for skincare
purposes. Our study examined the biological
effects of Douglas fir essential oil (DEO) in a human
skin disease model. The effects of DEO were
determined by measuring the levels of biomarkers
that are linked to inflammation, immune function,
and wound healing. The effects of DEO on
genome-wide gene expression were also studied.
DEO showed strong anti-proliferative and immune
modulatory activities. Notably, DEO affected critical
genes and pathways associated with immune
modulation and cancer signaling. The findings
from this study suggest that DEO is biologically
active in human skin cells and it potentially
modulates immune response and cancer biology.
Thus, this study provides an important stepping
stone for further research on DEO and its health
benefits in humans.
© 2017 Doterra International. This open access article is distributed under a Creative Commons
Attribution (CC-BY) 4.0 license.
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Han, Cogent Biology (2017), 3: 1336886
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Subjects: Biochemistry; Pharmaceutical Science; Pharmacology; Immunology
Keywords: Douglas fir essential oil; Pseudotsuga menziesii; beta-pinene; sabinene; terpinolene; immune modulation; anti-proliferation; anticancer; collagen; plasminogen activator
inhibitor 1
1. Introduction
Douglas fir (Pseudotsuga menziesii) essential oil (DEO) is typically composed of beta-pinene, sabinene, terpinolene, delta-3-carene, alpha-pinene, and several other aromatic substances in smaller
amounts. DEO has been reported to possess antimicrobial and antifungal properties (Johnston,
Karchesy, Constantine, & Craig, 2001; Tesevic et al., 2009). Although DEO has gained popularity in
skincare usage, scientific studies of its biological effects on human skin cells are limited. Therefore,
we sought to evaluate the biological activity of a commercially available DEO in a human fibroblast
model of chronic inflammation and fibrosis. In particular, we studied the effect of DEO on 17 important protein biomarkers that are critical for inflammation, immune modulation, and tissue remodeling. We also analyzed the impact of DEO on genome-wide gene expression profile. This study
provides the first evidence of biological activity of DEO in human skin cells. Our data suggest that
DEO may modulate immune responses and tumor signaling processes.
2. Materials and methods
All experiments were conducted in HDF3CGF, a biologically multiplexed activity profiling (BioMAP)
system, which includes a cell culture of human dermal fibroblasts designed to model chronic inflammation and fibrosis in a robust and reproducible way. The system consists of three components: a
cell type, stimuli to create the disease environment, and a set of biomarker (protein) readouts to
examine the effect of treatment on disease environment (Berg et al., 2010). The methodologies used
in this study are similar to those previously described (Han & Parker, 2017a, 2017b; Kunkel et al.,
2004).
2.1. Cell cultures
Primary human neonatal fibroblasts were prepared as previously described (Bergamini et al., 2012)
and were plated under low serum conditions for 24 h before stimulation with a mixture of interleukin
(IL)-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-ϒ, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF). The cell culture and stimulation conditions for the HDF3CGF assays have been described in detail elsewhere and were performed
in a 96-well plate (Bergamini et al., 2012; R Development Core Team, 2011).
2.2. Protein-based readouts
An enzyme-linked immunosorbent assay (ELISA) was used to measure the biomarker levels of cellassociated and cell membrane targets. Soluble factors in the supernatants were quantified using
either homogeneous time-resolved fluorescence detection, bead-based multiplex immunoassay, or
capture ELISA. The adverse effects of the test agents on cell proliferation and viability (cytotoxicity)
were measured using the sulforhodamine B (SRB) assay. For proliferation assays, the cells were cultured and quantified after 72 h (which is optimal for the HDF3CGF system), and the detailed procedure has been described in a previous study (Bergamini et al., 2012). Measurements were performed
in triplicate wells, and a glossary of the biomarkers used in this study is provided in Supplementary
Table S1.
Quantitative biomarker data are presented as the mean log10 relative expression level (compared
to the respective mean vehicle control value) ±standard deviation of triplicate measurements.
Differences in biomarker levels between DEO- and vehicle-treated cultures were tested for significance with the unpaired Student’s t-test. A p value <0.05 outside the significance envelope and with
an effect size of at least 10% (more than 0.05 log10 ratio units) was considered statistically
significant.
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2.3. RNA isolation
Total RNA was isolated from cell lysates using the Zymo Quick-RNA miniprep kit (Zymo Research
Corp., Irvine, CA, USA) according to the manufacturer’s instructions. RNA concentration was determined using a NanoDrop ND-2000 system (Thermo Fisher Scientific, Waltham, MA, USA). The RNA
quality was assessed using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and an
Agilent RNA 6000 Nano kit. All samples had a A260/A280 ratio between 1.9 and 2.1 and RNA integrity number score >8.0.
2.4. Microarray analysis for genome-wide gene expression
The effect of DEO (0.0037%, v/v) on the expression of 21,224 genes in the HDF3CGF system after 24 h
of treatment was examined. Samples for microarray analysis were processed by Asuragen Inc.
(Austin, TX, USA) according to the company’s standard operating procedures. Biotin-labeled cRNA
was prepared from 200 ng total RNA using an Illumina TotalPrep RNA amplification kit (Thermo
Fisher Scientific, Waltham, MA, USA) and one round of amplification. The cRNA yields were quantified
using ultraviolet spectrophotometry, and the distribution of the transcript sizes was assessed using
the Agilent Bioanalyzer 2100. Labeled cRNA (750 ng) was used to probe Illumina human HT-12 v4
expression bead chips (Illumina, Inc., San Diego, CA, USA). Hybridization, washing, staining with
streptavidin-conjugated cyanine-3, and scanning of the Illumina arrays were carried out according
to the manufacturer’s instructions. The Illumina BeadScan software was used to produce the data
files for each array; the raw data were extracted using Illumina BeadStudio software.
The raw data were uploaded into R (R Development Core Team, 2011) and analyzed for qualitycontrol metrics using the beadarray package (Dunning, Smith, Ritchie, & Tavare, 2007). The data
were normalized using quantile normalization (Bolstad, Irizarry, Astrand, & Speed, 2003), and then
re-annotated and filtered to remove probes that were non-specific or mapped to intronic or intragenic regions (Barbosa-Morais et al., 2010). The remaining probe sets comprised the data-set for the
remainder of the analysis. The fold-change expression for each set was calculated as the log2 ratio
of DEO to the vehicle control. These fold-change values were uploaded onto Ingenuity Pathway
Analysis (IPA, Qiagen, Redwood City, CA, USA, www.qiagen.com/ingenuity) to generate the networks
and pathway analyses.
2.5. Reagents
DEO (provided by dōTERRA, Pleasant Grove, UT, USA) was diluted in dimethyl sulfoxide (DMSO) to 8X
the specified concentrations (final DMSO concentration in culture media was no more than 0.1%
[v/v]); 25 μL of each 8X solution was added to the cell culture to a final volume of 200 μL. DMSO
(0.1%) served as the vehicle control. Chemical analysis of DEO by gas chromatography–mass spectrometry indicated that its major chemical constitutes (i.e. >5%) are beta-pinene (23%), sabinene
(17%), terpinolene (14%), delta-3-carene (11%) and alpha-pinene (9%).
3. Results and discussion
3.1. Biological activity of DEO in pre-inflamed human dermal fibroblasts
We analyzed the biological activity of DEO in the dermal fibroblast cell system HDF3CGF, which features the microenvironment of inflamed human skin cells with boosted inflammatory and immune
responses. DEO biological activity was assessed at four different concentrations (0.011, 0.0037,
0.0012, and 0.00041%, v/v). DEO was overly cytotoxic at 0.011% concentration, and thus, effects of
only three other concentrations were further analyzed. The expressions of several biomarkers were
significantly altered by the exposure of cells to 0.0037% DEO (Figure 1).
Furthermore, DEO showed significant anti-proliferative activity in dermal fibroblasts. DEO significantly inhibited increase in the production of vascular cell adhesion molecule 1 (VCAM-1), collagenIII, and plasminogen activator inhibitor 1 (PAI-1). VCAM-1 mediates adhesion of monocytes and T
cells to endothelial cells, and is considered as an inflammatory biomarker. Collagen-III is an extracellular matrix protein that belongs to the family of fibrillar collagen found in extensible connective
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Han, Cogent Biology (2017), 3: 1336886
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Figure 1. Bioactivity profile of
Douglas fir essential oil (DEO,
0.0037% v/v) in human dermal
fibroblast system HDF3CGF.
Notes: The X-axis indicates
protein biomarker readouts.
The Y-axis denotes log10
transformed relative
expression levels of biomarkers
following the exposure of cells
to DEO compared to the levels
observed in vehicle-treated
control cells. Vehicle control
values are shaded in gray,
denoting 95% confidence
level. Asterisks indicate
biomarker “key activity,” as
DEO-induced changes in
their expression levels were
significantly different (p < 0.05)
from those in vehicle-treated
controls with an effect size
of at least 10% (more than
0.05 log ratio units). MCP-1,
monocyte chemoattractant
protein; VCAM-1, vascular cell
adhesion molecule 1; ICAM1, intracellular cell adhesion
molecule 1; IP-10, interferon
gamma-induced protein 10;
I-TAC, interferon-inducible
T-cell alpha chemoattractant;
IL-8, interleukin-8; MIG,
monokine induced by gamma
interferon; EGFR, epidermal
growth factor receptor;
M-CSF, macrophage colonystimulating factor; MMP-1,
matrix metalloproteinase 1;
PAI-1, plasminogen activator
inhibitor 1; TIMP, tissue
inhibitor of metalloproteinase.
tissues. Collagen-III is involved in cell adhesion, cell migration, and tissue remodeling. PAI-1 is critically involved in tissue remodeling and fibrinolysis. The inhibitory effect of DEO on these protein
molecules suggests that DEO may possess immunomodulatory property, and thus, may affect
wound healing.
Terpinolene, a major active component of DEO, downregulates the expression of serine/threonine
protein kinase AKT1 in K562 cells and inhibits cell proliferation (Okumura, Yoshida, Nishimura,
Kitagishi, & Matsuda, 2012). Reports showed that terpinolene exhibited anticancer and antioxidative
effects in rat brain cells, and was therefore suggested to be a potent anti-proliferative agent for
brain tumor cells (Aydin, Türkez, & Taşdemir, 2013). More recently, the anti-inflammatory effect of
terpinolene has been demonstrated in a rat model of chronic inflammation (Macedo et al., 2016).
3.2. Effect of DEO on genome-wide gene expression
To better understand the effect of DEO on human cells, we studied the effect of 0.0037% DEO (the
highest tested concentration that was not overly cytotoxic to these cells) on RNA levels of 21,224
genes in the HDF3CGF system. We observed that DEO affected expression of a diverse set of human
genes. Among the 118 genes whose expression levels were significantly altered by DEO (with a log2
fold-change ratio of expression over vehicle control ≥|1.5|), the majority (81 out of 118 genes) were
significantly downregulated, whereas the rest were significantly upregulated (Table S2). A crosscomparison of the protein and gene expression data revealed that collagen III expression was inhibited by DEO at both protein and gene levels.
IPA showed that exposure to DEO significantly affected many canonical signaling pathways from
literature-validated databases (Figure 2). Many of these pathways are involved in cell cycle control,
cancer biology, and immune response. For example, the four most-matched pathways (and genes in
these pathways) were inhibited overall by DEO (Figure 2, Tables S3–S6). These findings indicate that
DEO might potentially modulate immune response and cancer biology.
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Figure 2. Top 20 canonical
pathways that match with the
profile of biological effects
of Douglas fir essential oil
(DEO, 0.0037%, v/v) on gene
expression in the HDF3CGF
system.
Notes: Calculations were
made using QIAGEN Ingenuity
Pathway Analysis. Each p
value, describing the likelihood
that the observed association
between a specific pathway
and the data-set is due to
random chance, was obtained
by using the right-tailed
Fisher’s exact test. Pathways
with smaller actual P value (or
conversely, bigger −ln [p-value],
indicated by black bars) match
more significantly with DEO
biological activity profile. The
ratios, indicated by gray bars,
were calculated by considering
the number of genes that
participate in a given canonical
pathway from the DEO dataset, and dividing it by the
total number of genes in that
pathway.
Several earlier studies have suggested that terpinolene might be a potential anticancer agent (Aydin
et al., 2013; Okumura et al., 2012). Studies of other essential oils that have relatively high content of
β-pinene and/or sabinene (which are also abundant in DEO) showed promising anticancer and immunomodulatory effects of these compounds (da Silva et al., 2016; Krifa et al., 2015; Sertel, Eichhorn, Plinkert,
& Efferth, 2011). Further research is required to explore the biological mechanisms of action of DEO.
The present study has several limitations. Although the disease model was designed to simulate
disease biology of chronic inflammation and fibrosis, the in vitro study results cannot be directly correlated with the more complex human system. The impact of DEO on gene expression was only
evaluated after short-term intervention. Therefore, the effect of DEO on genome-wide gene expression in longer term remains elusive. Nonetheless, the study provides evidence of the biological effect
of DEO on human skin cells based on protein and gene expression data, and will likely stimulate
further research into its mechanisms of action.
4. Conclusions
To the best of our knowledge, this is the first study to evaluate the biological activity of DEO in human
skin cell culture. DEO significantly inhibited cell proliferation and the production of VCAM-1, collagenIII, and PAI-1. Genome-wide gene expression profile changes indicated that DEO robustly impacted
genes and pathways that are critical for cancer signaling, DNA damage response, and immune modulation. These data suggest that DEO may modulate immune responses and cancer signaling. Further
investigations into the biological and physiological mechanisms of action of DEO are recommended.
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Han, Cogent Biology (2017), 3: 1336886
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Supplemental data
Supplemental data for this article can be accessed here
https://doi.org/10.1080/23312025.2017.1336886.
Acknowledgment
We thank Editage (www.editage.com) for English language
editing.
Funding
This study was funded by dōTERRA (Pleasant Grove, UT,
USA) and was conducted at DiscoverX (Fremont, CA, USA).
Competing Interests
Xuesheng Han is employee of dōTERRA, where the studied
compound DEO was manufactured.
Author details
Xuesheng Han1
E-mails: [email protected], [email protected]
ORCID ID: http://orcid.org/0000-0003-2720-3011
1
dōTERRA International, LLC, 389 S 1300 W, Pleasant Grove,
UT 84062, USA.
Citation information
Cite this article as: In vitro biological activities of Douglas
fir essential oil in a human skin disease model, Xuesheng
Han, Cogent Biology (2017), 3: 1336886.
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